The therapeutic treatment of diseases and disorders by gene therapy involves the transfer and stable insertion of new genetic information into cells. The correction of a genetic defect by re-introduction of the normal allele of a gene encoding the desired function has demonstrated that this concept is clinically feasible [Rosenberg et al. (1990) New Eng. J. Med., 323, 570].
Hematopoietic stem cells or pluripotent progenitor cells are particularly useful for gene therapy studies since, although they are somatic cells, they differentiate to produce all the lineages of blood cells. Hence, the introduction of a foreign gene into a stem or progenitor cell results in the production of various lineages which can potentially express the foreign gene or alter control of native gene products. The introduction of a foreign gene into a progenitor cell or any other appropriate cell requires a method of gene transfer to integrate the foreign gene into the cellular genome. Although a variety of physical and chemical methods have been developed for introducing exogenous DNA into eukaryotic cells, viruses have generally been proven to be much more efficient for this purpose. Several DNA-containing viruses such as parvoviruses, adenoviruses, herpesviruses and poxviruses, and RNA-containing viruses, such as retroviruses, have been used to develop eukaryotic cloning and expression vectors. The fundamental problem with retroviruses is that they are either the etiologic agents of, or are intimately associated with, malignancy. Retroviruses integrate randomly into the cellular genome, and thus may activate cellular proto-oncogenes or may disrupt sequences critical to cell function. Accordingly, the use of retroviral vectors in gene transfer presents a problem in that there is a finite chance that such vectors may induce neoplasia. Thus, a need exists for additional and improved vectors for gene transfer.
Whereas retroviruses are frequently the etiologic agents of malignant disorders, parvoviruses constitute the sole group of DNA-containing viruses that have not yet been associated with any malignant disease. Although parvoviruses are frequently pathogenic in animals, a parvovirus of human origin, the adeno-associated virus 2 (AAV), has so far not been associated with any known human disease, even though up to 90% of the human population has been exposed to AAV. [Blacklow, N.R. (1988) in: Parvoviruses and Human Disease, CRC Press, Boca Raton). In addition, most retroviruses used for gene transfer are of murine origin, while AAV, a human virus, is physiologically more relevant for gene transfer in humans. Moreover, retroviruses are susceptible to inactivation by heat and organic solvents, whereas AAV is heat stable, extremely resistant to lipid solvents, and stable between pH 3.0 and 9.0. Thus as vehicles for gene transfer, parvoviruses provide many advantages over retroviruses.
Recombinant retroviruses have low viral titers (10.sup.5 -10.sup.6 virions/ml) (Rosenberg) in contrast to the high titers of recombinant AAV (10.sup.8 -10.sup.9 virions/ml) [Srivastava et al. (1990) Blood 76, 1997]. Consequently, it is generally not possible to achieve an infection efficiency with recombinant retrovirus beyond 10-50% of the target cell population, with successful infection requiring actively replicating cells. In contrast, a 70% infection efficiency has been reported for a recombinant AAV [Samulski et al. (1989) J. Virol. 63, 3822], and it is possible to achieve a 100% infectivity of target cells with wild-type AAV [Nahreini et al. (1989) Intervirol. 30, 74]. Furthermore, even though recombinant retroviral vectors have been rendered replication-incompetent, there remains a low probability of recombination between the vector and endogenous retroviral sequences. In contrast, 60-90% of the population is sero-positive for human parvoviruses, and no endogenous viral sequences have yet to be detected in volunteer donors. In recombinant AAV vectors, all of the AAV coding sequences have, nonetheless, been deleted.
Perhaps the most significant advantages of AAV-based vectors are that they mediate integration into the host chromosomal DNA in a site-specific and stable manner. Retroviral genomes, following reverse transcription, undergo integration into the host chromosomal DNA with a totally random integration pattern. AAV establishes a latent infection which is site-specific. The integration site has been mapped to human chromosome 19. (Kotin et al. (1990) Proc. Natl. Acad. Sci. USA 87, 2211). It has therefore become feasible to accomplish site-specific delivery of exogenous DNA into mammalian cells. While retroviral vectors mediate integration of non-viral sequences into the host chromosome, the integration pattern is not always stable. Frequently the integrated retroviral provirus is excised from the cell. AAV, on the other hand, establishes a stable integration.
Despite the potential advantages outlined above, the parvovirus-based vectors suffer from one limitation, and that is the size of a DNA sequence that can be packaged into the mature virions. For example, whereas up to 8.0-9.0 kilobase pair (kbp) DNA fragments can be packaged into retroviral vectors, a maximum of about 5.0 kbp DNA can be packaged into AAV. This size limitation, however, does not preclude the cloning and packaging of most cDNA molecules.
Thus parvovirus-based vectors offer a useful alternative to retroviral vectors for gene therapy in humans. While AAV-based vectors allow stable, site-specific integration of transferred genes, the indiscriminate expression of the transferred gene in all cell lineages presents significant problems. Thus, a need exists for AAV vectors which effect tissue-specific expression of the transferred gene. In accordance with the present invention, one method, for example, to solve this problem is by a combination of the features of AAV and another human parvovirus, B19.
While AAV causes no known disease, B19 is known to be the etiologic agent of a variety of clinical disorders in humans. B19 is the causative agent of transient aplastic crises associated with various hemolytic anemias, erythema infectiosum or the "fifth disease", post-infection polyarthralgia and thrombocytopenia in adults, and some cases of chronic bone marrow failure and hydrops fetalis.
AAV is dependent on a helper virus, such as adenovirus, herpesvirus, or vaccinia virus, for optimal replication. In the absence of a helper virus, AAV establishes a latent infection in which the viral genome integrates into chromosomal DNA site-specifically. B19, on the other hand, is an autonomously replicating virus that is known to replicate only in human hematopoietic cells in the erythroid lineage. Both AAV and B19 contain linear, single-stranded DNA genomes, but their genomes show no homology at the nucleotide sequence level. The nucleotide sequences of both genomes are known. [Lusby et al. (1980) J. Virol. 34, 402; Srivastava et al. (1983) J. Virol. 45, 555; Shade et al. (1986) J. Virol. 58, 921]. The AAV genome contains inverted terminal repeats (ITRs) of 145 nucleotides, 125 nucleotides of which form a palindromic hairpin that plays a critical role during AAV DNA replication. The sequences of the ITRs are shown in FIG. 1 and as SEQ ID NO:1. In latently infected cells, the termini of AAV are at the junction of the cellular sequences and thus the termini also facilitate integration and rescue.
The remarkable features of the two human parvoviruses can be combined, for example, in an AAV-B19 hybrid vector, to provide vectors in accordance with the present invention. The vectors of this invention are particularly useful for gene transfer in bone marrow cells and other hematopoietic cells. These hybrid viral vectors mediate site-specific integration as well as tissue-specific expression of heterologous genes in hematopoietic cells.